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Abstract

Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.

rAAV.CHOPS2053.GFP transduces cone photoreceptors in the squirrel monkey. Near the injection site, which was placed just inferior to the fovea, GFP fluorescence was observed within cones. At the near peripheral location of the field shown, the cone density is high and there are relatively few rods. At this eccentricity from the fovea in the primate retina, rods and cones are easily distinguishable because of marked anatomical differences, with cone inner segments being 2 to 3 times wider in diameter than rods. At a more peripheral location distal to the injection site (inset), cones are even more distinct with their larger diameters, and fewer cells were transduced as evidenced by isolated cones expressing GFP surrounded by nontransduced cells. In the near periphery, it appears that a majority of cones express GFP. Within the same field, there is also an occasional cell with a narrow profile which expresses GFP. This may be spurious expression of GFP in rods. In locations distal from the injection site where occasional GFP expressing cones are observed, no suspect rods expressing GFP were seen. Abbreviations: OS=outer segment; IS=inner segment; ONL=outer nuclear layer.

GFP fluorescence imaged in a living monkey using the RetCam II. All images show the right eye of animal 265750. Images in panels (A)–(D) were obtained using a halogen bulb; images in panels (E)–(H) were obtained using a xenon bulb. In panels (D)–(H), images were obtained with a green filter (510nm cutoff) placed between the light source and the camera for detecting GFP fluorescence. (A) Fundus image taken immediately before the injection procedure. Blood vessels provide landmarks that allow comparisons to be made across images. (B) Same retinal area as panel (A), which corresponds to the location of the injection that was made inferior and temporal to the optic nerve head, imaged directly following the injection procedure. A large bleb of virus-containing solution that was made underneath the retina is visible. The bleb and retinal landmarks are labeled in (C). (D) Image taken 9
weeks postinjection using a 130° lens. (E) Same retinal area as panel D showing a small, roughly J-shaped area of GFP expression. (F) Image taken 12
weeks postinjection using a high-magnification 30° lens. The smaller lens was found to prevent unfiltered light at the edges, thus eliminating the greenish background seen in the image shown in panel (E). In images (F)–(H), all of the green light is coming strictly from the GFP fluorescence. (G) Same retinal area shown in panel (F), imaged at 20
weeks postinjection. (H) An image taken at 24
weeks postinjection. (I) Graph of the relative intensity of the GFP fluorescence in images (E)–(H). Pixel intensities are plotted on a scale in which the lowest possible intensity equals zero and the highest equals 100.